This project focuses on glycoconjugates of Schwann cells and oligodendrocytes during myelination and demyelination. A major aspect of the research concerns the myelin-associated glycoprotein (MAG), which is localized in periaxonal glial membranes of myelinated fibers and functions in transmitting signals between axons and myelin-forming cells. MAG is in the "siglec" subgroup of the immunoglobulin superfamily and binds to glycoconjugates containing terminal alpha2-3-linked sialic acid, suggesting that its axonal receptor or ligand could be a glycoprotein or ganglioside. Much has been learned about the function of MAG from investigation of a line of MAG-null mice, which had been generated on a mixed background. In the past year, we completed 10 generations of backcrossing with C57BL/6J mice to obtain a congenic line of MAG-null mice exhibiting 99.5% C57BL/6J-derived genome content. This congenic line should substantially facilitate research on MAG. [unreadable] [unreadable] In the PNS, MAG signaling from Schwann cells to axons is essential for the normal maintenance of myelinated axons. In the absence of MAG, the pathology in the PNS is characterized by degeneration of myelinated axons. This is preceded by a reduction of axonal caliber caused by cytoskeletal abnormalities including decreased expression and phosphorylation of neurofilaments, due in part to decreased activities of extracellular signal regulated kinases 1 & 2 (ERK 1/2) and cyclin dependent kinase 5 (cdk5). The well-known role of MAG as one of several white matter inhibitors of neurite outgrowth also shows that MAG is able to influence the properties of axons. However it is unclear how this capacity to inhibit the growth of plastic developing or regenerating neurites relates to its normal function in glia-axon interactions within the periaxonal region of myelinated axons. The capacity to inhibit outgrowth may be an early manifestation of a MAG-mediated signaling system that promotes axonal maturation to eventually optimize their structure for rapid conduction of action potentials in mature myelinated axons. In the past few years, research in many laboratories on the inhibition of neurite outgrowth has provided much new information about a neuronal receptor for MAG, which appears to involve a complex of the Nogo receptor (NgR), the p75 neurotrophin receptor (p75NtR), other proteins and gangliosides. A principal goal of our ongoing research is to determine how these findings with immature plastic neurons relate to MAG-mediated signaling within the periaxonal compartment of myelinated axons that is essential for their normal maintenance. Recent experiments involve Western blotting with a membrane fraction isolated from white matter that is enriched in axolemma and periaxonal oligodendroglial membranes. MAG is enriched in this fraction, whereas the NgR and p75NtR are present, but not enriched. Levels of the NgR and p75NtR in the fraction are higher in developing brain than adult brain. [unreadable] [unreadable] An important function of MAG in the CNS appears to be signaling in the opposite direction from axons to oligodendrocytes to promote their differentiation and survival. Morphological studies on the CNS of MAG-null mice revealed a significant delay of myelination, aberrant or redundant myelin loops and abnormal paranodal structures. Thus in the absence of MAG, some oligodendrocytes do not seem to be efficient at determining when, where and how much myelin to form. Furthermore, there is a loss of oligodendroglial proteins and degeneration of periaxonal oligodendroglial processes in aging MAG-null mice consistent with a "dying back" oligodendrogliopathy, similar to that occurring in some demyelinating lesions of multiple sclerosis. These findings suggest that MAG functions as the receptor for an axonal signal that enhances the process of myelination and the vitality of oligodendrocytes. Experiments are in progress with primary oligodendrocyte cultures and oligodendroglial cell lines to demonstrate and characterize a MAG-mediated signaling pathway that promotes the differentiation and/or survival of these cells. One approach for activating MAG-mediated signaling in cultured oligodendrocytes is cross-linking with anti-MAG antibodies. Under control conditions, little oligodendroglial MAG is associated with lipid rafts, but upon activation, much of the MAG shifts into the raft fraction. This is associated with a number of changes in the oligodendroctyes, including activation of Fyn tyrosine kinase, dephosphorylation of serine and threonine residues in some other proteins, and cleavage of alpha-fodrin followed by a transient depolymerization of actin. More recent experiments have shown that MAG-mediated signaling also activates oligodendroglial ERK 1/2. These in vitro results support the hypothesis that MAG is a receptor for transmitting an axonal signal that activates a signaling cascade affecting the properties of oligodendrocytes